Bottom Line:
Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay.DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci.These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.

ABSTRACTControl of the rearrangement and expression of the T cell receptor alpha and delta chains is critical for determining T cell type. The process of delta deletion is a candidate mechanism for maintaining separation of the alpha and delta loci. Mice harboring a transgenic reporter delta deletion construct show alpha/beta T cell lineage-specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic delta-deleting elements now in gamma/delta T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.

Figure 3: Schematic representation of the δ deletion transgenic constructs. (a) Map of TG1. The vertical bars represent cloning sites in the construction of TG1 (21). (b) Magnification of the 1.9-kb SalI–XhoI fragment of TG1. The black box represents the 48-bp segment HPS1A, the region of DNA deleted to make TG2.

Mentions:
Results from TG1 strongly suggested that regulatory signal sequences around δREC, if they exist, must be contained within the relatively small 1.9-kb DNA segment containing human δREC (Fig. 3 a; 21). Because of its proximity to the h-s-n and its temporal restriction to early T cells, HPS1A became the prime candidate for a region controlling lineage-specific use of δ-deleting elements. In an attempt to determine if HPS1A is important in the δ deletion process, TG2 was assembled using oligonucleotide-directed site-specific mutagenesis. The 1.9-kb SalI–XhoI δREC-containing fragment in Fig. 3 b was mutated to delete the 48-bp segment, HPS1A (black box). The newly generated 1.85-kb mutant fragment was ligated into the base construct (Fig. 3 a) to make TG2. TG2 was injected by the University of Alabama at Birmingham (Birmingham, AL) Transgenic Facility and seven founder lines containing the construct were created. All seven lines had expected transgenic bands by Southern analysis, and ranged in copy number from 2 to 20 copies (data not shown). The estimated copy number of each transgene is indicated in Table 2.

Figure 3: Schematic representation of the δ deletion transgenic constructs. (a) Map of TG1. The vertical bars represent cloning sites in the construction of TG1 (21). (b) Magnification of the 1.9-kb SalI–XhoI fragment of TG1. The black box represents the 48-bp segment HPS1A, the region of DNA deleted to make TG2.

Mentions:
Results from TG1 strongly suggested that regulatory signal sequences around δREC, if they exist, must be contained within the relatively small 1.9-kb DNA segment containing human δREC (Fig. 3 a; 21). Because of its proximity to the h-s-n and its temporal restriction to early T cells, HPS1A became the prime candidate for a region controlling lineage-specific use of δ-deleting elements. In an attempt to determine if HPS1A is important in the δ deletion process, TG2 was assembled using oligonucleotide-directed site-specific mutagenesis. The 1.9-kb SalI–XhoI δREC-containing fragment in Fig. 3 b was mutated to delete the 48-bp segment, HPS1A (black box). The newly generated 1.85-kb mutant fragment was ligated into the base construct (Fig. 3 a) to make TG2. TG2 was injected by the University of Alabama at Birmingham (Birmingham, AL) Transgenic Facility and seven founder lines containing the construct were created. All seven lines had expected transgenic bands by Southern analysis, and ranged in copy number from 2 to 20 copies (data not shown). The estimated copy number of each transgene is indicated in Table 2.

Bottom Line:
Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay.DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci.These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.

ABSTRACTControl of the rearrangement and expression of the T cell receptor alpha and delta chains is critical for determining T cell type. The process of delta deletion is a candidate mechanism for maintaining separation of the alpha and delta loci. Mice harboring a transgenic reporter delta deletion construct show alpha/beta T cell lineage-specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic delta-deleting elements now in gamma/delta T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.